PROJECT SUMMARY Acute myeloid leukemia (AML) is susceptible to immunotherapy as evidenced by the success of allogeneic (allo) stem cell transplantation (SCT) in this disease. Although allo-SCT can be curative in leukemia, it carries a high rate of treatment-related mortality and morbidity. This is primarily a result of off-target immune responses elicited by donor-derived cytotoxic T cells (CTL) within the SCT graft against normal tissues, a phenomenon known as graft-versus-host disease (GvHD), which occurs in up to 50% of patients following allo-SCT. Furthermore, AML relapse remains the leading cause of mortality following allo-SCT, highlighting the shortfalls of allo-SCT in providing long-lasting cures. In order to minimize GvHD, while taking advantage of the graft versus leukemia (GvL) effect, numerous leukemia-associated antigens (LAAs) have been identified and shown to elicit leukemia-specific immune responses. PR1 is an HLA-A2-restricted LAA that we identified in our lab and targeted using a monoclonal T cell receptor (TCR)-like antibody (8F4), a PR1-peptide vaccine, and PR1- CTL. In the current proposal, we plan to engineer chimeric antigen receptor (CAR) T cells that target PR1/HLA- A2 on the surface of AML using the 8F4 construct. The rationale for this proposal is that in view of the shortcomings and significant toxicities associated with allo-SCT, balanced by the susceptibility of AML to immunotherapy (i.e. allo-SCT), there is a critical need to develop novel immunotherapies to achieve disease elimination with minimal off-target toxicity. We plan to use 8F4 as the CAR since it has a very high affinity for the PR1/HLAA2 epitope presented by AML. We plan to use cord blood T cells as the cell source for engineering the 8F4-CAR T cells due to the success we have recently encountered in engineering and expanding sufficient numbers of effective 8F4-CAR T cells using cord blood products. Furthermore, we showed potency of the cord blood-derived 8F4-CAR-T cells in treating human AML in a mouse xenograft model. We will also introduce a caspase 9 (iCP9) suicide gene into the CAR construct, to increase the safety profile of the 8F4-CAR-T cells. Our central hypothesis is that immunotherapy with iCP9-8F4-CAR T cells engineered from cord blood T cells will eliminate PR1-expressing AML, with minimal off-target toxicity. We will (1) validate the safety and anti-leukemic activity of the iCP9-8F4-CAR-T cells in animal models using primary patient AML samples and cell lines; (2) test the safety and efficacy of the iCP9-8F4-CAR-T cells in patients with AML as a bridge to allo-SCT; and (3) study immune reconstitution and perform correlative studies using blood and bone marrow samples from iCP9-8F4-CAR-T cell recipients. After completion of our proposed studies, we anticipate that PR1-targeting adoptive cellular therapy using 8F4-CAR-T cells could become a standard therapy for patients with myeloid leukemia. In addition, our studies will elucidate the potential for cord blood T cells in the engineering of CAR-T cells for the treatment of cancer.